Evolution and function analysis of auxin response factors reveal the molecular basis of the developed root system of Zygophyllum xanthoxylum

Background As a xerophytic shrub, forming developed root system dominated with lateral roots is one of the effective strategies for Zygophyllum xanthoxylum to adapt to desert habitat. However, the molecular mechanism of lateral root formation in Z. xanthoxylum is still unclear. Auxin response factors (ARFs) are a master family of transcription factors (TFs) in auxin-mediated biological processes including root growth and development. Results Here, to determine the relationship between ARFs and root system formation in Z. xanthoxylum, a total of 30 potential ZxARF genes were first identified, and their classifications, evolutionary relationships, duplication events and conserved domains were characterized. 107 ARF protein sequences from alga to higher plant species including Z. xanthoxylum are split into A, B, and C 3 Clades, consisting with previous studies. The comparative analysis of ARFs between xerophytes and mesophytes showed that A-ARFs of xerophytes expanded considerably more than that of mesophytes. Furthermore, in this Clade, ZxARF5b and ZxARF8b have lost the important B3 DNA-binding domain partly and completely, suggesting both two proteins may be more functional in activating transcription by dimerization with AUX/IAA repressors. qRT-PCR results showed that all A-ZxARFs are high expressed in the roots of Z. xanthoxylum, and they were significantly induced by drought stress. Among these A-ZxARFs, the over-expression assay showed that ZxARF7c and ZxARF7d play positive roles in lateral root formation. Conclusion This study provided the first comprehensive overview of ZxARFs and highlighted the importance of A-ZxARFs in the lateral root development. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04717-7.


Introduction
Roots are the frontline organs in contact with environmental signals in soil, and play essential roles in whole plant growth and development.The root system of higher plants consists of an embryonic primary root and postembryonic lateral roots and adventitious roots.In dicot plants, lateral roots are remarkably plastic in adapting to ever-changing growth conditions, which is vital for expanding root system to absorb water and nutrients, and even to survival [1].
It is well known that auxin plays essential roles in almost all stages of root development [2,3].Previous studies about the model plant Arabidopsis thaliana (Arabidopsis) have identified a major auxin signaling pathway consisting of transport inhibitor response 1/ auxin signaling F-box protein (TIR1/AFB1-5) receptors, auxin/indole-3-aceticacid (Aux/IAA) repressors and auxin response factor (ARF) transcription factors (TFs) [4,5].As the central players of auxin signaling pathway, ARFs regulate plant growth and development by regulating the expression of early/primary auxin response genes [4].
Since AtARF1 was first identified from Arabidopsis in 1997 [6], ARF gene families of many species have been characterized.For instance, 3 ARFs in Marchantia polymorpha [7], 23 ARFs in Arabidopsis [8], 19 ARFs in Punica granatum [9], 19 ARFs in Citrus sinensis [10], 39 ARFs in Litchi chinensis [11], and 40 ARFs in Medicago truncatula [12] have been identified through genomewide data mining.Despite undergoing hundreds of millions of years of evolution, ARF families still maintain unique conserved domains across plants [13,14].A typical ARF protein is characterized by a highly conserved N-terminal DNA-binding domain (DBD) that includes a B3 domain and an ARF domain, a middle region (MR) with variable sequences dividing the TFs as an activator or repressor, and two conserved C-terminal domains (CTD) [4,13].The two CTDs form a Phox and Bem1p (PB1) domain, which often confers interaction specificity in highly redundant protein scaffolds to facilitate signaling events [4].This domain also presents in most of the Aux/IAA proteins, and thus named AUX/IAA domain [4].As transcription factors, ARFs are located in the nucleus and their localization signals are in DBDs.
According to the phylogenetic analysis and predicts, ARF proteins are divided into A (A-ARFs, ARF5/6/7/8/19), B (B-ARFs, ARF1/2/3/4/9) and C (C-ARFs, ARF10/16/17) Clades with diverse roles in plants [13], which have been extensively detailed by studying on Arabidopsis.A-ARFs, as a class of activated TFs, played important roles in the process of lateral root and adventitious root development in Arabidopsis [15,16].The single loss-of-function mutants arf5, arf6 and arf7 and the double mutants arf6 arf8 and arf7 arf19 all showed different degrees of defects in lateral root and adventitious root development [16][17][18][19].Particularly, the arf7 arf19 double knockout mutant was severely impaired in lateral root formation [17].For B-ARFs, studies have revealed that the knockout mutant of ARF3 produced severe defects in carpel development, and arf3 arf4 double mutant showed a breakdown of abaxial tissue specification in all lateral organs [20].In addition, several C-ARFs positively regulated the formation of nitrogen-fixing nodule by regulating the balance of auxin and cytokinin in legumes [21].Arabidopsis ARF10/16 played key roles in the differentiation of root-stem cells [22].Moreover, there are some ARF genes that seem to be involved in plant responses to stresses.For instance, phosphorylation of ARF2 modulated the expression of the K + transporter gene HAK5 (high affinity K + transporter 5) in response to low potassium stress in Arabidopsis [23].SUMO (small ubiquitin-like modifier)-dependent regulation of ARF7 controlled root branching pattern in response to water availability [24].Chinese cherry (Cerasus pseudocerasus) CpARF7 participated in root development and responded to drought and low phosphorus stresses [25].
As mentioned above, most of these studies have focused on mesophytes, whereas ARFs in desert xerophytes with special strategies and excellent tolerance to environmental stresses remain largely uncharacterized.As a succulent xerophyte, Zygophyllum xanthoxylum has a developed root system dominated with robust primary root as well as well-developed lateral and adventitious roots.Especially, developed lateral roots confers Z. xanthoxylum strong ability for absorbing water and nutrients, which is one of the effective strategies to adapt to the drought and barren desert habitat [26,27].However, the molecular mechanism of lateral root formation in Z. xanthoxylum is elusive.
Given that identification of gene families from distinct model plants is a necessary step in formulating better hypotheses related to physiological and developmental characteristics [28], in this study, ZxARF gene family were firstly identified on the basis of the whole genome sequences of Z. xanthoxylum (PRJNA933961), and comparative evolution analyses of ZxARF and ARF protein sequences of some representative species were presented to elucidate their evolution and function related to plant growth and development.In addition, the role of ZxARFs in lateral root development was further confirmed by over-expression assay in Arabidopsis.

ZxARF genes were identified in Z. xanthoxylum genome
We firstly isolated the complete arrays of ZxARF proteins by using the 23 ARF sequences of Arabidopsis (AtARF) as queries to blast the database of the annotated Z. xanthoxylum genome.After redundant result elimination and further conserved domain validation, a total of 30 potential ZxARF genes were finally identified and were named as detailed in Table 1 according to their homologues in Arabidopsis (Table 1).
Detailed information of the predicted 30 ZxARF genes were further analyzed.The results showed that ORF length of these ZxARF genes range from 1110 bp (ZxARF17a) to 3360 bp (ZxARF7a) (Table 1), and the numbers of exon of these genes range from 2 (ZxARF16b, 17a, 17b) to 15 (ZxARF5a, 6a).(Table 1).Correspondingly, the length and molecular weight vary significantly among these ZxARF proteins.ZxARF7a is the largest protein containing 1119 amino acids, whereas ZxARF17a is the smallest one, which is consist of 369 amino acids (Table 1).And the molecular weights of these proteins range from 40.28 kDa (ZxARF17a) to 123.14 kDa (ZxARF7a) (Table 1).In addition, the theoretical pI values of ZxARFs range from 5.16 (ZxARF5c) to 8.31 (ZxARF2a).The pI values of 23 ZxARFs are smaller than 7, indicating they are acidic protein, and the rest of 7 ZxARFs are alkaline (pI > 7) (Table 1).All the data collected suggested a high variability among the ZxARF genes in the Z. xanthoxylum genome.Furthermore, Plant-mPLoc software was used to predict the probable protein localization of ZxARFs.As TFs, all of ZxARFs contain a nucleus-targeting signal (Table 1), suggesting they are nuclear localization proteins.

Phylogenetic analysis of ZxARFs and AtARFs
Phylogenetic analysis plays an important role in functional predictions of various genes across species [29,30].To study the phylogenetic relationships between the members of ZxARF and AtARF genes, an unrooted phylogenetic tree was constructed from an alignment of the corresponding protein sequences and viewed in MEGA-X program by the maximum likelihood method (Fig. 1).It was showed that the phylogenetic tree fell into three broad groups (  C. Interestingly, the ratio of ZxARFs and AtARFs in Clade A was 2.4:1.This result is consistent with the developed root system of Z. xanthoxylum, since A-ARFs positively regulate lateral root development [15].Also, the number of C-ZxARFs is two fold of C-AtARFs, which may be associate with the root development of Z. xanthoxylum according to the previous study of Arabidopsis [22].

Evolution analysis of ARF proteins
The origin and adaptive evolution are vital to predict the function of genes [31].To elucidate the phylogenetic relationships and evolutionary history of plant ARF genes, the phylogenetic tree was constructed by a maximum likelihood method, based on 107 ARF protein sequences from alga to higher plant species, including Chlorokybus atmophyticus (1), Chara braunii (1), M. polymorpha (3), Physcomitrella patens (13), Selaginella moellendorfii (7), Ginkgo biloba (15), Amborella trichopoda (14), Arabidopsis (23) and Z. xanthoxylum (30) (Fig. 2a, b).ARF family members in C. braunii and A. trichopoda were firstly identified in this study (Fig. 2b).Meanwhile, except that the members of ARFs in S. moellendorfii are only 7, which is different from the reports of Jo et al. [32], the else are in line with previous studies [7,8,14,33,34] (Fig. 2b).Phylogenetic analysis showed that these ARF proteins were also split into three Clades: Clade A-C.ARF proteins of the 2 chlorophytes were only located in Clade C, whereas ARF proteins of bryophytes were distributed in Clade A-C.These results indicated that Clade C probably be the origin of ARF proteins in land plants, as well as that ARF proteins in Clade A and B may play vital roles in the evolution of land plants.The phylogenetic tree also showed that CaARF is the most ancient ARF protein, which is in line with the previous study [14].ARF family members had expanded largely in the process of evolution.Among them, there were no A-ARF members in the rootless algae, while the number of A-ZxARFs was the largest among the plants (Fig. 2b and Table 2).It further demonstrates that the expansion of A-ZxARFs may be an important reason for the formation of the developed root system in Z. xanthoxylum.

Comparative analysis of ARF proteins between xerophytes and mesophytes
For additionally evidencing that A-ZxARFs expansion is related to the formation of developed root system of Z. xanthoxylum, another unrooted phylogenetic tree was constructed based on ARF protein sequences from diploid xerophytes (Z.xanthoxylum and Pugionium dolabratum) and diploid mesophytes (Camellia.sinensis, Ci. sinensis, and Arabidopsis) (Fig. 3a, b).All these plants shared an ancient (130-140 Mya) triploidization event [35], and Z. xanthoxylum, P. dolabratum and Ca.sinensis underwent another independent whole-genome duplication (WGD) event in subsequent evolution, respectively [36,37].In addition, while Ca.Sinensis and Ci.sinensis are also shrubs and their shoot sizes are similar to that of Z. xanthoxylum, their root systems are less developed than that of Z. xanthoxylum [27,38,39].Arabidopsis is the best studied model plant.Thus, these plants are suitable for comparative analysis to explore the relationship between the evolution of A-ARFs and root development.
Here, the ARF family members of Ca. sinensis (25) were further complemented base on the study of Xu et al. [40].And the ARF family members of P. dolabratum were firstly identified in this study.ARFs in Ci. sinensis were obtained from Li et al. [10].We found that these ARFs were also clustered into A-C 3 Clades (Fig. 3b).The number of A-ARFs from Z. xanthoxylum, P. granatum, Arabidopsis, Ca. sinensis, and Ci.Sinensis were 12, 11, 5, 6, and 6, respectively (Fig. 3b and Table 3), which were consistent with the root characteristics of these plants [27,36,38,39].

Analysis of chromosomal localization and gene replication events of ZxARFs
The distribution of ZxARFs on chromosomes were mapped based on the genomic database.Expect for 2 genes lie within unassembled, 28 ZxARFs are located on 11 chromosomes of Z. xanthoxylum randomly (Fig. 4a).Five ZxARFs are located on chromosome 3, three on chromosomes 1, 2, 6 and 11, two on chromosomes 4, 5, 7, 8 and 10, and one on chromosome 9, respectively (Fig. 4a).
To further infer the phylogenetic mechanisms and potential gene duplication events of ZxARFs, the synteny relationships and Ks value among the orthologous ZxARF genes were performed (Fig. 4b and Table 4).The Ks value of the first and second WGD of Z. xanthoxylum range from 0.80-1.16and 0.22-0.52,respectively.Our results indicated that most of the ZxARF genes were amplified by WGD or chromosomal fragment duplication events (Fig. 4).By comparing the Ks values between the orthologous ZxARFs gene pairs and WGD of Z. xanthoxylum, it was found that the ancestral genes of ZxARF7c and ZxARF7d were generated by the ancient WGD, and ZxARF5b, ZxARF6b and ZxARF7d were generated by the recent WGD (Table 4).

Expression levels of A-ZxARFs in Z. xanthoxylum root systems and their expression analysis in response to osmotic stress
Expression pattern of a gene is correlated with its function.To verify whether these A-ZxARFs were involved in the growth and development of root, their expressions b Phylogenetic analysis of ARF family members of plants in a. ARF amino acids sequences were used for constructing an unrooted tree using the maximum likelihood method with 1000 bootstrap replications by MEGA-X.In total, 107 ARFs were analyzed and divided into 3 Clades (A-C), which were exhibited in red, green, and orange, respectively.Distinctive species and the corresponding proteins are indicated with different colored symbols as Fig. 2a in Z. xanthoxylum root system were confirmed by RT-qPCR.As shown in Fig. 5a, the Δct value of almost all A-ZxARFs were less than 10, indicating that they have high expression levels in the Z. xanthoxylum root system.Among them, the highest expressed gene was ZxARF5b and the lowest expressed gene was ZxARF7c (Fig. 5a).
Extensive research has shown that various environmental signals are integrated into changes in auxin homeostasis, redistribution, and signaling [41].To further confirm the function of A-ZxARF genes, we analyzed their expression levels under osmotic stress treatment (simulate drought stress) (Fig. 5b).Almost all A-ZxARFs were induced by osmotic stress treatment and peaked at 30 min (Fig. 5b).Among them, the expression level of ZxARF5b, ZxARF7c, ZxARF6c and ZxARF7d showed the most significant changes, with 13.58, 8.57, 6.35 and 5.40-fold up-regulation at 30 min, respectively (Fig. 5b).Therefore, we hypothesized that drought stress signals may induce the expression of A-ZxARFs to regulate root development of Z. xanthoxylum, resulting in the formation of the developed root system.

Protein domain analysis of ZxARFs
Protein structure is vital to determine the function of a protein and its interactive network.Thus, domains of ZxARF proteins were identified and analyzed using NCBI Conserved Domain Database.The results showed that most of the ZxARFs shared similar domain compositions in the same groups (Fig. 6), suggesting functional similarities within the same subfamily.

ZxARF7c and ZxARF7d promote LR growth and development
AtARF7/19 plays a key role in LR formation in Arabidopsis, and ZxARF7c and ZxARF7d are highly homologous to AtARF7/19 (Fig. 1).To characterize the function of ZxARF7c and ZxARF7d in LR growth and development, the coding sequence (CDS) of ZxARF7c and ZxARF7d were cloned.Stabilized ZxARF7c-OE lines (Line a and Line b) and ZxARF7d-OE lines (Line 1 and Line 2) were generated by overexpressing ZxARF7c and ZxARF7d under a 35S promoter in the arf7 arf19 double mutant of Arabidopsis, respectively.RT-PCR assay confirmed that ZxARF7c and ZxARF7d transcripts were obviously increased in ZxARF7c-OE and ZxARF7d-OE lines and no detectable in arf7 arf19 double mutant seedling (Fig. S1-S3).The results showed that defect of LR development of arf7 arf19 seedlings are rescued completely by ZxARF7c and ZxARF7d overexpression, respectively (Figs. 7 and 8).Similarly, the rosette of arf7 arf19 adult plants were rescued via ZxARF7c and ZxARF7d overexpression, respectively (Figs. 9 and 10).
In addition, the transgenic plants had larger root surface area and root volume, more root forks and longer total root length than the arf7 arf19 background plant (Figs.9d-h and 10d-h).Also, fresh weight and dry weight of the root from these transgenic plants are higher compared to that of arf7 arf19 mutant (Figs.9b-c and 10bc).These collective data indicates that ZxARF7c and ZxARF7d play a positive role in LR formation and growth.

Discussion
It has been reported that ARF gene family play vital roles in root development and response to abiotic stresses [23][24][25].However, the functions of xerophytes ARF genes are remained poorly characterized.In the present study, we first identified 30 ZxARF proteins from the recent genome sequencing of the desert xerophytes Z. xanthoxylum (Table 1).Furthermore, a comprehensive information of ZxARF gene family has been provided and analyzed.
ARF family are divided into A, B, and C Clades in land plants [13].Finet et al. [13] inferred that Clade A and C can probably be traced back to the origin of land plant, since they include bryophyte sequences.However, Kato et al. [7] have identified a B-ARF from M. polymorpha.Meantime, Martin-Arevalillo et al. [14] found that the most 'ancient' ARF homologue is C. atmophyticus ARF (CaARF), which belongs to Clade C. Similarly, in this study, we identified that the only one ARF protein from C. braunii is also a C-ARF (Fig. 2b).Therefore, we may conclude that the first ARF proteins originated from algae, which probably be a C-ARF.As a super-xerophyte, Z. xanthoxylum has formed highly developed root system network dominated by lateral roots [27].The root systems of mesophytes, such as Ca.sinensis and Ci.sinensis, are less developed [27,38,39].By constructing phylogenetic tree of ARFs from Z. xanthoxylum and these mesophytes including Ca. sinensis and Ci.sinensis at the same evolutionary stage (Fig. 3b), it is noteworthy that the number of A-ZxARFs is far greater than that of A-ARFs from these mesophytes.In addition, as another super-xerophyte, P. dolabratum also harbours as many A-ARFs numbers ARF amino acids sequences were used for constructing an unrooted tree using the maximum likelihood method with 1000 bootstrap replications by MEGA-X.In total, 128 ARFs were analyzed and divided into 3 Clades (A-C), which were exhibited in red, green, and orange, respectively.Distinctive species and the corresponding proteins are indicated with different colored symbols as Fig. 3a as Z. xanthoxylum (Fig. 3b).Given that A-AtARFs play vital roles in every stage of lateral roots development in Arabidopsis [15], we concluded that A-ARFs from other plants also play important roles in the lateral roots development, and the massive expansion of A-ZxARFs may be the important mechanism for the formation of the developed root system of the xerophyte Z. xanthoxylum.
In addition, based on the fossil evidence and the root anatomy of extant vascular plants, three root-evolution events were proposed during vascular plant evolution, giving rise to the extant bifurcating roots in lycophytes, adventitious/lateral roots in euphyllophytes (ferns and seed plants), and primary roots in seed plants [42].According to the phylogenetic tree of ARFs from alga to higher plant species, we found that ARF10/16 in Clade C is restricted to phanerogams.Given that ARF10/16 are involved in the root development in Arabidopsis [22], we speculate that these proteins of ARF10/16 branch may play important roles in root evolution in seed plants rather than lycophytes.
A typical ARF protein contains three domains, designated as DBD, MR and PB1 [4].Most ZxARFs were characterized by the classic domains, while 11 members lacked the PB1 domain and 3 members lacked the typical B3 DNA-binding domains (Fig. 6).Especially, ZxARF5b and ZxARF8b (A-ZxARFs) have lost the B3 domain partly and completely (Fig. 6).Ulmasov et al. [5] found that ARFs with Q-Rich MRs can activate transcription on TGT CTC AuxREs in the absence of a DBD.Similarly, Tiwari et al. [43] found that the ARF MR can function as an AD or an RD when fused to a heterologous DBD, and targeted to non-auxin response gene promoters.Therefore, ZxARF5b and ZxARF8b may function as activators and compete with other A-ZxARFs to bind the transcriptional repressor ZxAux/IAAs, which help release these A-ZxARFs from ZxAux/IAAs-ZxARFs dimers to trigger transcription of downstream genes, and then initiate lateral root formation.Due to ZxARF5c only has a truncated ARF domain, it might be a pseudogene, or express and function at specific developmental stages under special conditions.The exact contributions of these ARFs remain to be elucidated, especially for those that lack one or even two domains.

Conclusions
In this study, we found that A-ZxARFs had expanded largely in the process of evolution, which may be the important mechanism for the formation of the developed root system of Z. xanthoxylum.This assumption was further confirmed by over-expression assay in Arabidopsis.Our studies will pave the way for further understanding the potential role of A-ZxARF genes in plant root development.

Plant materials and growth conditions
Seeds of Z. xanthoxylum were collected from the Minqin (38°03′N, 101°49′E; elevation 1371 m) region, which were identified by Minqin desert botanical garden (http:// www.nfgrp.cn/ data/ list/ resou rce_ detai llist.html), and stored in the Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Afairs, Lanzhou, China.The corresponding voucher specimen (Chase 1700 (K)) has been deposited in stored in School of Life Science, Shihezi University [44].Z. xanthoxylum plants were grown under long-day conditions (16 h/8 h light/dark; 600 μmol m −2 s −1 light intensity; 40%-65% relative humidity; 28 ± 2 ℃) for 3 weeks.For the drought treatment, irrigation was 1/2 Hoagland solution containing 300 mM Mannitol.Control (CK) plants were all irrigated with 1/2 Hoagland solution.Roots were sampled after 15 min, 30 min 1 h and 3 h treatment.All samples were immediately frozen in liquid nitrogen after harvesting and stored at -80 °C until use.
The T-DNA insertion alleles arf7 arf19 (CS24630) was obtained from the Arabidopsis Biological Resource Center (ABRC), and was identified and propagated by our group before [16].For plate assay, Arabidopsis seeds were placed on 1/2 Murashige and Skoog (MS) medium containing 1% (w/v) sucrose and cold-treated at 4℃ for 4 d, and then transferred to long-day conditions (16 h/8 h light/dark; 100 μmol m −2 s −1 light intensity; 40%-65% relative humidity; 22℃ ± 2℃) to grow for 10 d [16].For soil culture assay, Arabidopsis seeds were cold-treated at 4℃ for 4 d, and then sown in sterilized soils and grown in long-day conditions as above.

Identification of plant ARF family members
The annotated protein sequences of C. braunii, S. moellendorfii, A. trichopoda and Ca.sinensis were all obtained from National Center for Biotechnology Information (NCBI, https:// www.ncbi.nlm.nih.gov/) database resources.The annotated protein sequences of P. dolabratum were obtained from Hu et al. [36].The annotated  Twenty-three ARF amino acids sequences of Arabidopsis were used as queries against protein database of C. braunii, S. moellendorfii, A. trichopoda, Ca.Sinensis, P. dolabratum and Z. xanthoxylum, respectively.The possible ARF proteins were further characterized by BLAST analysis via TAIR database.Finally, all identified ARFs were further validated by a conserved domain search using the CDD (http:// www.ncbi.nlm.nih.gov/ cdd/) and Pfam (http:// pfam.sanger.ac.uk/) databases.Candidate proteins without ARF domain were removed.All ARF   S1.

Establishment of phylogenetic trees for plants and ARF proteins
Based on ANGIOSPERM PHYLOGENY WEBSITE (https:// www.mobot.org/ MOBOT/ resea rch/ APweb/), we established plant phylogenetic trees of the representative species in plant evolutionary history including C. atmophyticus, C. braunii, M. polymorpha, S. moellendorfii, P. patens, G. biloba, A. trichopoda, Arabidopsis and Z. xanthoxylum.Also, we established plant phylogenetic trees of the mesophytic and xerophytic species including Arabidopsis, Ca.Sinensis, Ci.Sinensis, P. dolabratum and Z. xanthoxylum.Phylogenetic analysis of ARF proteins were carried out according to the maximum likelihood method using the Clustal W and MEGA X software packages, and support for each node was assessed by performing a bootstrap analysis with 1000 replicates [45].

Quantitative real-time PCR analysis
An RNAprep Pure Plant Kit (Tiangen) was used to isolate total RNA of leaves from each plant and firststrand cDNA was synthesized from the RNA by using a PrimeScript ™ RT Master Mix Kit (TaKaRa).qRT-PCR was performed in triplicate on three bioreplicates using Power SYBR ™ Green Master Mix (TaKaRa Biotechnology, China) on a StepOne Real-Time PCR Thermocycler (Applied Biosystems).All kits were used according to the manufacturer instructions.Primer-BLAST (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast) was used for primer design according to the following criteria: PCR amplicon lengths of 80-120 bp, Tm of 60 ± 1℃ and GC contents of 45-60%.The primers employed were listed in Table S2.The comparative cycle threshold method (ΔΔCT) was used to calculate relative expression levels, and ZxACTIN (GenBank: EU019550.1)from Z. xanthoxylum was used as the reference gene.Agrobacterium-mediated gene transfer under arf7 arf19 backgrounds [47].Transgenic plants were selected by 0.01% BASTA [16].The expressions of 35S::ZxARF7c-FLAG and 35S::ZxARF7d-FLAG were confirmed with semi-quantitative reverse-transcription-polymerase chain reaction (RT-PCR).And two independent transgenic lines of each gene were used for further analyses, respectively.Primers for gene clones were listed in Table S2.

Semi-quantitative reverse-transcription-polymerase chain reaction
An RNAprep Pure Plant Kit (Tiangen) was used to isolate total RNA of leaves from each plant and firststrand cDNA was synthesized from the RNA by using a PrimeScript ™ RT Master Mix Kit (TaKaRa) according to the manufacturer's instructions.AtACTIN (TAIR: At3g18780) was used as the reference gene.
Semi-quantitative PCR was performed in 18 cycles for AtACTIN2 and 35 cycles for ZxARF7c and ZxARF7d.The PCR products were analyzed on 2% agarose gels.Equal loading of each amplified gene sequence was determined with the AtACTIN2 PCR product.All amplifications were performed at least three times.Primers for RT-PCR assay were listed in Table S2.

Root system traits measurements
Arabidopsis seedlings grown on 1/2 MS solid medium for 10 d.Length of the primary roots was measured by Image J (Version 1.42q, NIH) software.Number of LRP/ LR was counted with an Olympus light microscope.The density of LRP/LR was indicated via dividing LRP/ LR number by the primary root length.The number of each replicate for per genotype was carried out at least 10 plants.For 3-week-old Arabidopsis plants grown in pots, the roots were gently washed to separate and then placed on a transparent tray filled with water.The root trays were scanned with an Epson Perfection V700 scanner to obtain a grayscale TIFF image.Subsequently, the images were analyzed by using the WinRHIZO Pro image processing system (Regent Instruments Inc., 2672 Chemin Sainte-Foy, Quebec City, Quebec G1V 1 V4, Canada) to obtain root surface area, root volume, root forks, and total root length.Next, roots were dried with absorbent paper to get their fresh weight, and to obtain their dry weight after being placed at 75℃ for 48 h.The number of replicates per genotype was carried out at least 6 plants.All photographs were taken with a digital camera.

Fig. 1
Fig. 1 Phylogenetic tree analysis of ARFs from Z. xanthoxylum and Arabidopsis.In total, 30 ARFs of Z. xanthoxylum and 23 ARFs of Arabidopsis were used for constructing an unrooted tree via the maximum likelihood method with 1000 bootstrap replications by MEGA-X.These ARFs divided into 3 Clades (A-C), which were exhibited in red, blue, and green, respectively.The red solid circles represent ZxARFs and the black solid triangles represent AtARFs

Fig. 2
Fig. 2 Phylogenetic trees of plants and their ARF family members.a Phylogenetic trees of the representative species in plant evolutionary history.The evolution analysis of C. atmophyticus, C. braunii, M. polymorpha, P. patens, S.moellendorfii, G. biloba, A. trichopoda, Arabidopsis, and Z. xanthoxylum were performed using the ANGIOSPERM PHYLOGENY WEBSITE.The solid triangles represent spore plants and the solid circles represent seed plants.bPhylogenetic analysis of ARF family members of plants in a. ARF amino acids sequences were used for constructing an unrooted tree using the maximum likelihood method with 1000 bootstrap replications by MEGA-X.In total, 107 ARFs were analyzed and divided into 3 Clades (A-C), which were exhibited in red, green, and orange, respectively.Distinctive species and the corresponding proteins are indicated with different colored symbols as Fig.2a

Fig. 3
Fig. 3 Phylogenetic trees of 3 mesophytes and 2 xerophytes and their ARF family members.a Phylogenetic trees of 3 mesophytes and 2 xerophytes.The evolution analysis of Z. xanthoxylum, P. dolabratum, Ci. sinensis, Ca.Sinensis and Arabidopsis were performed using the ANGIOSPERM PHYLOGENY WEBSITE.Distinctive species are indicated with different colored solid triangles.b Phylogenetic analysis of ARF family members of plants in a.ARF amino acids sequences were used for constructing an unrooted tree using the maximum likelihood method with 1000 bootstrap replications by MEGA-X.In total, 128 ARFs were analyzed and divided into 3 Clades (A-C), which were exhibited in red, green, and orange, respectively.Distinctive species and the corresponding proteins are indicated with different colored symbols as Fig.3a

Fig. 4
Fig. 4 Chromosomal location and syntenic analyses of ZxARFs.a Chromosomal location of ZxARFs.The chromosomal position of each ZxARF gene was mapped according to the Z. xanthoxylum genome.The chromosome number was indicated at the left side of each chromosome using black characters.The ZxARFs gene number was indicated at the right side of each chromosome using red characters.The scale bar represents the length of the chromosomes, and the units is in megabases (Mb).b Syntenic analyses of ZxARFs.The syntenic relationships of ARF genes in Z.xanthoxylum were built with MCScanX program by TBtools.The putative orthologous ZxARF genes were presented in brown.The chromosome numbers and gene names are indicated in and around the circle, respectively

Fig. 5
Fig. 5 Expression analysis of A-ZxARFs in Z. xanthoxylum root.a Relative transcript abundance profiles of A-ZxARFs in roots of Z. xanthoxylum.The individual gene names are indicated at the bottom.Different letters on the bars indicate significant difference (P < 0.05; n > 3 plants per column).b Expression pattern analysis of A-ZxARFs in roots of Z. xanthoxylum under osmotic stress (Simulated drought) treatment.Different colors in map represent different transcript abundance values.The scale bar on the right indicates relative expression levels.Time of osmotic stress treatments are indicated at the bottom.The individual gene names are indicated on the right

Fig. 6
Fig.6 Analyses of phylogenetic relationships and conserved domains of ZxARFs.30 ARFs of Z. xanthoxylum were used for constructing an unrooted tree using the maximum likelihood method with 1000 bootstrap replications by MEGA-X.These ZxARFs divided into 3 Clades (A-C), which were exhibited in red, blue, and green, respectively.Lengths of each domain are displayed proportionally.The blue boxes represent B3 domains, green boxes represent ARF domains, and purple boxes represent PB1 domains.The blue and purple solid circle represents the absence of B3 domain and PB1 domain, respectively

Fig. 7
Fig. 7 Root phenotype analysis of 35S::ZxARF7c-FLAG in arf7/19-related seedlings.a Phenotypes of arf7/19 and ZxARF7c-FLAG overexpression lines grow in light for 10 d after germination.Scale bar = 1 cm; b-d Statistical analysis of PRs length (b) and LRs and LRPs number (c) and density of LRs and LRPs (d) of plants in a. Different letters on the bars indicate significant difference (P < 0.05; n > 10 plants per column)

Fig. 8
Fig. 8 Root phenotype analysis of 35S::ZxARF7d-FLAG in arf7/19-related seedlings.a Phenotypes of arf7/19 and ZxARF7d-FLAG overexpression lines grow in light for 10 d after germination.Scale bar = 1 cm; b-d Statistical analysis of PRs length (b) and LRs and LRPs number (c) and density of LRs and LRPs (d) of plants in a. Different letters on the bars indicate significant difference (P < 0.05; n > 10 plants per column)

Fig. 9
Fig. 9 Phenotypes analysis of 35S::ZxARF7c-FLAG in arf7/19-related seedlings.a Phenotypes of rosette.Scale bar = 1 cm; b Phenotypes of root system.Scale bar = 1 cm; c-f Statistical analysis of root fresh weight (c) and root dry weight (d) and root surface area (e) and root volume (f) and root forks (g) and total length of root system (h) of plants in b.Different letters on the bars indicate significant difference (P < 0.05; n > 10 plants per column)

Fig. 10
Fig. 10 Phenotypes analysis of 35S::ZxARF7d-FLAG in arf7/19-related seedlings.a Phenotypes of rosette.Scale bar = 1 cm; b Phenotypes of root system.Scale bar = 1 cm; c-f Statistical analysis of root fresh weight (c) and root dry weight (d) and root surface area (e) and root volume (f) and root forks (g) and total length of root system (h) of plants in b.Different letters on the bars indicate significant difference (P < 0.05; n > 10 plants per column)

Table 1
Clade A, B, and C) with well-supported bootstrap values.There were 12 ZxARFs and 5 AtARFs in Clade A, 12 ZxARFs and 15 AtARFs in Clade B, 6 ZxARFs and 3 AtARFs in Clade The protein characteristics of ZxARFs in Z.

Table 2
The number of plant ARF family proteins

Table 4
Ks values of orthologous ZxARF gene pairs